CN108139392B

CN108139392B - Method for measuring PIVKA-II and method for producing PIVKA-II immunoassay reagent or kit

Info

Publication number: CN108139392B
Application number: CN201680058383.5A
Authority: CN
Inventors: 北村由之; 青柳克己
Original assignee: Fujirebio Inc; Sekisui Medical Co Ltd
Current assignee: Fujirebio Inc; Sekisui Medical Co Ltd
Priority date: 2015-10-07
Filing date: 2016-10-06
Publication date: 2020-05-08
Anticipated expiration: 2036-10-06
Also published as: EP3361252A1; WO2017061546A1; KR102549704B1; US10830771B2; KR20180055823A; EP3361252A4; JP6713478B2; CN108139392A; US20180284119A1; JPWO2017061546A1

Abstract

Disclosed is a means which enables the construction of a high-precision PIVKA-II immunological assay system using a monoclonal antibody against human prothrombin. The method for measuring PIVKA-II of the present invention comprises measuring PIVKA-II in a subject by immunoassay. The immunoassay uses a mixture of a 1 st anti-prothrombin antibody or antigen-binding fragment thereof recognizing a hydrophilic PIVKA-II molecule and a 2 nd anti-prothrombin antibody or antigen-binding fragment thereof recognizing a hydrophobic PIVKA-II molecule, and an anti-PIVKA-II antibody or antigen-binding fragment thereof specifically binding to PIVKA-II.

Description

Method for measuring PIVKA-II and method for producing PIVKA-II immunoassay reagent or kit

Technical Field

The present invention relates to a PIVKA-II measurement method capable of detecting PIVKA-II molecules contained in a sample without omission, and a method for producing a PIVKA-II measurement reagent or a kit.

Background

PIVKA-II (Protein induced by vitamin K absence-II, vitamin K deficiency induced Protein-II) is a glycoprotein with a structure similar to prothrombin associated with blood coagulation. Prothrombin is a 579-residue protein, and 10 glutamic acid (Glu) residues located near the N-terminus are γ -carboxylated to form γ -carboxyglutamic acid (Gla) residues. This N-terminal region is referred to as Glu-Gla region. When prothrombin is produced in vivo, it is known that gamma-carboxylation is incomplete due to deficiency of vitamin K, liver failure, administration of a vitamin K antagonist, liver cell damage, and the like, and glycoproteins in which all or a part of 10 residues form a Glu residue appear in blood. The protein is PIVKA-II, also known as abnormal prothrombin. In recent years, it has been reported that PIVKA-II is detected at a high concentration in plasma of hepatocellular carcinoma patients, and has been used as a marker for hepatocellular carcinoma in the monitoring of diagnosis. In addition to the Glu-Gla region, prothrombin is not structurally different from PIVKA-II, and any protein has 2 kringle domains (prothrombin fragment 1(F1) region, prothrombin fragment 2(F2) region) in the central region and a thrombin region at the C-terminal end.

As a method for specifically detecting PIVKA-II in a subject, the following immunoassay methods have been reported: both a monoclonal antibody that specifically recognizes PIVKA-II and a polyclonal antibody against prothrombin were used, and one of them was measured as an immobilized antibody and the other was measured as a labeled antibody (patent document 1). However, polyclonal antibodies can vary in specificity and affinity for prothrombin due to batch variation. To avoid this situation. Multiple batches need to be evaluated for specificity and affinity, and in order to ensure higher specificity, it is more desirable to utilize monoclonal antibodies rather than polyclonal antibodies.

In addition, in the measurement of PIVKA-II by ELISA, when a monoclonal antibody that specifically recognizes PIVKA-II is used as a solid-phase antibody and a polyclonal antibody against prothrombin is used as a secondary antibody, it is known that the secondary antibody contains an antibody that is reactive with thrombin, and thus a measurement system of a serum specimen is adversely affected, and a stable measurement value cannot be obtained (patent document 2). Patent document 2 reports: in order to solve this problem, an antibody that does not react with human thrombin but specifically reacts with human prothrombin is used as a secondary antibody, whereby the serum specimen can be stabilized and PIVKA-II can be measured. However, this method also uses a polyclonal antibody, and therefore has the same problem as patent document 1. Further, in the method of patent document 2, in order to remove an antibody against thrombin from a polyclonal antibody against human prothrombin, it is necessary to obtain an antibody reactive with prothrombin using a human prothrombin affinity column and then dialyze the antibody, and further to obtain an antibody non-reactive with thrombin using a human thrombin affinity column and dialyze the antibody, which requires a very complicated purification step of the antibody.

As another example of the PIVKA-II assay technique using ELISA, the following technique is reported: PIVKA-II was measured using a mixture of an anti-PIVKA-II monoclonal antibody as a solid-phase antibody and an anti-F1 monoclonal antibody and an anti-F2 monoclonal antibody as marker antibodies (patent document 3). According to this technique, the correlation between serum and plasma of a pair of samples of serum and plasma collected from the same patient at substantially the same time can be improved. However, in patent document 3, improvement of detection accuracy of PIVKA-II itself is not regarded as a technical problem.

Furthermore, the Glu-Gla region of PIVKA-II in blood has been analyzed in detail mainly about the decarboxylated site, and it is suggested that the region is rich in molecular diversity. However, the molecular diversity of PIVKA-II from the tricyclic domain to the C-terminal side other than the Glu-Gla region has not been investigated so far, and none of the above patent documents 1 to 3 is described.

Documents of the prior art

Patent document

Patent document 1, Japanese patent laid-open No. Sho 60-60557

Patent document 2, Japanese patent laid-open publication No. 5-249108

Patent document 3, Japanese patent laid-open publication No. 2014-35278

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide a means by which an immunoassay system for PIVKA-II can be constructed with higher accuracy using a monoclonal antibody against human prothrombin.

Means for solving the problems

The present inventors have conducted extensive studies on an immunoassay system for PIVKA-II using an anti-human prothrombin monoclonal antibody, and as a result, they have found that, depending on the combination of a subject and an anti-human prothrombin monoclonal antibody, the measurement results may not be integrated, and it is considered that there is a possibility that a subject (patient) whose PIVKA-II cannot be accurately measured by the conventional technique may exist.

As a result of further intensive studies, it was found that PIVKA-II molecules have not only molecular diversity in the Glu-Gla region but also a plurality of molecular species having different degrees of hydrophobicity. The content ratio of the plurality of PIVKA-II molecules having different degrees of hydrophobicity does not vary depending on the type of the subject (whether plasma or serum), and for example, the content ratio of PIVKA-II molecules having different degrees of hydrophobicity varies among subjects having serum. It has further become clear that: there are many known anti-human prothrombin monoclonal antibodies, but these monoclonal antibodies have different affinities for different levels of hydrophobicity of PIVKA-II molecular species, and thus there are antibodies having a high affinity for a highly hydrophobic molecular species and antibodies having a high affinity for a less hydrophobic molecular species. For example, it has been clarified that: when an antibody recognizing a highly hydrophobic molecular species is used in an immunological measurement method for PIVKA-II, most of PIVKA-II contained in a sample containing a large amount of a highly hydrophilic molecular species is not detected, and thus PIVKA-II in blood cannot be accurately measured.

The present inventors have found that PIVKA-II can be measured with high accuracy by measuring PIVKA-II using a mixture of an antibody having affinity for a PIVKA-II molecular species having high hydrophobicity and an antibody having affinity for a PIVKA-II molecular species having low hydrophobicity when constructing a PIVKA-II measurement system using a monoclonal antibody recognizing a prothrombin region, and have completed the present invention.

That is, the present invention provides a method for measuring PIVKA-II, which comprises measuring PIVKA-II in a subject by immunoassay using a mixture of at least 1 st anti-prothrombin antibody or antigen-binding fragment thereof that recognizes a hydrophilic PIVKA-II molecule and at least 1 nd anti-prothrombin antibody or antigen-binding fragment thereof that recognizes a hydrophobic PIVKA-II molecule, and an anti-PIVKA-II antibody or antigen-binding fragment thereof that specifically binds to PIVKA-II. The present invention also provides a method for producing a PIVKA-II immunoassay reagent or kit, comprising: a step of examining the reactivity of an antibody or an antigen-binding fragment thereof that binds to both PIVKA-II and prothrombin but does not exhibit reactivity with thrombin, with respect to a hydrophilic fraction and a hydrophobic fraction of PIVKA-II; and a step of mixing at least 1 antibody or an antigen-binding fragment thereof that reacts with the hydrophilic fraction and at least 1 antibody or an antigen-binding fragment thereof that reacts with the hydrophobic fraction.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, the PIVKA-II molecule group contained in the blood specimen can be measured with high accuracy. The present inventors have found for the first time that PIVKA-II molecules in blood are a collection of molecules having different degrees of hydrophobicity, and the proportion of PIVKA-II molecules having high hydrophobicity to PIVKA-II molecules having low hydrophobicity differs for each sample. Conventional immunoassay methods using a monoclonal antibody against prothrombin have not been able to accurately detect the PIVKA-II group in blood because of the nature of the antibody used, without carefully adjusting the hydrophobicity/hydrophilicity level of the PIVKA-II molecule in blood. According to the method of the present invention, a plurality of PIVKA-II molecular species which could be missed in the conventional method can be detected with high accuracy, and thus the accuracy of PIVKA-II measurement can be dramatically improved.

Drawings

FIG. 1 shows the results of measurement of PIVKA-II molecules in a sample obtained by fractionating 6 samples of serum samples derived from hepatocellular carcinoma patients by hydrophobic interaction chromatography, using an ALP-labeled antithrombin polyclonal antibody as an enzyme-labeled antibody.

FIG. 2-1 shows the results of measurement of PIVKA-II molecules in a sample (No.4) derived from a hepatocellular carcinoma patient by subjecting the individual fractions to hydrophobic interaction chromatography as measurement samples and using labeled antibodies A to D.

FIG. 2-2 shows the results of measurement of PIVKA-II molecules in a sample obtained by fractionating a serum specimen (No.269) derived from a hepatocellular carcinoma patient by hydrophobic interaction chromatography using labeled antibodies A to D as measurement samples.

FIG. 2-3 shows the results of measurement of PIVKA-II molecules in a sample (No.275) derived from a serum specimen of a hepatocellular carcinoma patient by subjecting the respective fractions to hydrophobic interaction chromatography as measurement samples using labeled antibodies A to D.

FIG. 3-1 shows the results of PIVKA-II in a sample obtained by fractionating a serum specimen (No.4) derived from a hepatocellular carcinoma patient by hydrophobic interaction chromatography, using the respective fractions as measurement samples, and using antibody mixtures (antibody C/A, antibody C/B, and antibody C/D) as labeled antibodies.

FIG. 3-2 shows the results of measurement of PIVKA-II in a sample obtained by fractionating a serum specimen (No.269) derived from a hepatocellular carcinoma patient by hydrophobic interaction chromatography using an antibody mixture (antibody C/A, antibody C/B, and antibody C/D) as a labeled antibody.

FIG. 3-3 shows the results of measurement of PIVKA-II in a sample obtained by fractionating a serum specimen (No.275) derived from a hepatocellular carcinoma patient by hydrophobic interaction chromatography using an antibody mixture (antibody C/A, antibody C/B, and antibody C/D) as a labeled antibody as a measurement sample.

Fig. 4-1 shows the results of measuring PIVKA-II in a sample using, as a labeled antibody, an antibody mixture obtained by mixing antibodies C/a at various weight ratios (a: C: 1: 30:1) as measurement samples for respective fractions obtained by fractionating a serum specimen (No.275) derived from a hepatocellular carcinoma patient by hydrophobic interaction chromatography.

Fig. 4-2 shows the results of measuring PIVKA-II in a sample using, as a labeled antibody, an antibody mixture obtained by mixing antibodies C/a at various weight ratios (a: C: 3:1 to 0.03:1) as measurement samples for respective fractions obtained by fractionating a serum specimen (No.275) derived from a hepatocellular carcinoma patient by hydrophobic interaction chromatography.

Detailed Description

The method for assaying PIVKA-II of the present invention is a method for immunologically assaying PIVKA-II using an antibody or an antigen-binding fragment thereof that binds to PIVKA-II, and a mixture of at least 1 antithrombin antibody or an antigen-binding fragment thereof that recognizes a hydrophilic (low-hydrophobicity) PIVKA-II molecule and at least 1 antithrombin antibody or an antigen-binding fragment thereof that recognizes a hydrophobic (high-hydrophobicity) PIVKA-II molecule is used as one of the antibody or the antigen-binding fragment thereof. In the present invention, for convenience, the former antibody recognizing a hydrophilic PIVKA-II molecule is referred to as "antibody 1", and the latter antibody recognizing a hydrophobic PIVKA-II molecule is referred to as "antibody 2".

The present inventors have found that PIVKA-II molecules having different degrees of hydrophobicity are present in a sample. It is presumed that the difference in hydrophobicity of the PIVKA-II molecule is not caused by the degree of γ -carboxylation in the Glu-Gla region, but the diversity of the three-loop region in the center of the molecule and the structure located on the C-terminal side thereof is caused by the structural diversity.

The antithrombin antibody is: an antibody that recognizes and binds both PIVKA-II and prothrombin but does not exhibit reactivity with thrombin. The phrase "not exhibiting reactivity" as used herein means that: does not bind to thrombin at detectable levels (i.e., binds to thrombin below background); alternatively, even if binding is detected at detectable levels, the degree of binding is significantly less than that to PIVKA-II and prothrombin, and binding is only determined by those skilled in the art to be non-binding to thrombin. Antithrombin antibodies are known per se and commercially available. The 1 st and 2 nd anti-prothrombin antibodies used in the present invention may be selected from known anti-prothrombin antibodies, or they may be newly prepared and selected from anti-prothrombin antibodies. The details of the selection methods for the 1 st and 2 nd anti-prothrombin antibodies will be described later.

Further, as another antibody or an antigen-binding fragment thereof, an anti-PIVKA-II antibody or an antigen-binding fragment thereof that specifically binds PIVKA-II while distinguishing PIVKA-II from prothrombin is used. Specificity of anti-PIVKA-II antibodies can be expressed as "binding PIVKA-II only and showing no reactivity with prothrombin". The meaning of "not showing reactivity" is as described above. The anti-PIVKA-II antibody recognizes a Glu-Gla region, which is a region characteristic of PIVKA-II and is not present in prothrombin, and binds to PIVKA-II molecules regardless of hydrophobicity or hydrophilicity.

Such an anti-PIVKA-II antibody is also known (for example, patent document 1), and there are also commercially available products. Alternatively, a newly prepared anti-PIVKA-II antibody can be used.

The 1 st and 2 nd anti-prothrombin antibodies and the anti-PIVKA-II antibody may be polyclonal or monoclonal, and monoclonal antibodies are preferred from the viewpoint of reproducibility of immunoassay and the like.

Methods for producing antibodies and antigen-binding fragments thereof are well known per se and conventional methods are known. The anti-prothrombin polyclonal antibody can be obtained, for example, as follows: an animal (excluding human) is immunized with prothrombin or a partial fragment thereof (partial fragment excluding Glu-Gla region) together with an appropriate adjuvant, antiserum is obtained from blood collected from the animal, and a polyclonal antibody in the antiserum is purified. In order to raise the antibody titer in the immunized animal, immunization usually needs to be performed several times within several weeks. The antibody in the antiserum can be purified by, for example, ammonium sulfate precipitation, fractionation by anion chromatography, affinity column purification, or the like.

The antithrombin monoclonal antibody can be prepared, for example, by a known hybridoma method. Specifically, an animal (excluding humans) is immunized with prothrombin or a partial fragment thereof (partial fragment other than the Glu-Gla region) together with an appropriate adjuvant, antibody-producing cells such as spleen cells and lymphocytes are collected from the animal, and fused with myeloma cells to prepare hybridomas, and the hybridomas that produce antibodies that bind to prothrombin are selected and proliferated, and the anticoagulant monoclonal antibody can be obtained from the culture supernatant. In the selection of hybridomas, PIVKA-II can be used as an antigen, or binding to both prothrombin and PIVKA-II can be confirmed.

An anti-PIVKA-II antibody can be prepared using a partial fragment of Glu-Gla region whose carboxylation is incomplete as an immunogen, and in the present invention, a monoclonal antibody is generally used because it is required to have sufficient specificity for PIVKA-II. With regard to the PIVKA-II monoclonal antibody, after a hybridoma is prepared using PIVKA-II or a partial fragment thereof (a partial fragment containing at least a part of 10 residues of the Glu-Gla region subjected to carboxylation) as an immunogen, a hybridoma producing an antibody that binds to PIVKA-II but does not bind to prothrombin is selected, and the antibody is recovered. Specific examples of the method for producing an anti-PIVKA-II antibody include the methods described in Japanese patent application laid-open Nos. 60-60557 and 9-249699.

The "antigenic binding fragment" may be any antibody fragment as long as it retains the binding property (antigen-antibody reactivity) to the corresponding antigen of the original antibody. Specific examples thereof include Fab and F (ab')₂scFv, etc., but not limited thereto. As is well known, Fab or F (ab')₂The monoclonal antibody can be obtained by treating it with a proteolytic enzyme such as papain or pepsin. scFv (Single chain fragment of variable region)Single-chain antibody), for example, a method for preparing a single-chain cDNA by extracting mRNA of the hybridoma prepared as described above, amplifying an immunoglobulin H chain gene and an immunoglobulin L chain gene by PCR using primers specific to the immunoglobulin H chain and the immunoglobulin L chain, ligating them with a linker, providing appropriate restriction enzyme sites to the amplified genes, introducing the amplified genes into a plasmid vector, transforming escherichia coli with the vector, expressing scFv, and recovering scFv from escherichia coli.

The polypeptide or a partial fragment thereof to be used as an immunogen can be produced by a conventional method such as chemical synthesis or genetic engineering. Alternatively, prothrombin and PIVKA-II can be extracted from fresh human plasma or the like and purified (see Thromb. diet. Haemorph.1966; 16:469-90, etc.).

The amino acid sequence shown in the sequence number 1 of the sequence table is an amino acid sequence with a signal sequence added on the prothrombin and PIVKA-II sequences, wherein the 44 th to 622 th amino acid regions correspond to the prothrombin and PIVKA-II amino acid sequences. The amino acid region from position 44 to 84 is a Glu-Gla region, and in prothrombin, all 10 "Xaa" in this region are γ -carboxyglutamic acid (Gla) residues. The prothrombin sequence is registered in GenBank under accession number NP _ 000497. The nucleotide sequence shown in SEQ ID NO.2 is a sequence encoding the amino acid sequence of SEQ ID NO. 1 and is registered in GenBank under accession number NM-000506. The region from base 44 to base 1912 in SEQ ID NO.2 is a coding region.

Specific examples of the chemical synthesis method include: fmoc method (fluorenylmethyloxycarbonyl method), tBoc method (t-butoxycarbonyl method), and the like. Alternatively, the peptide can be synthesized by a conventional method using various commercially available peptide synthesizers. In chemical synthesis, the desired polypeptide can be synthesized based on only the amino acid sequence.

Methods for making polypeptides by genetic engineering methods are also well known. In the case of producing an antithrombin antibody, a polypeptide to be used as an immunogen can be produced by a genetic engineering method, for example, as follows. First, RNA is extracted from cultured human cells or the like, and cDNA is synthesized from mRNA by reverse transcription reaction. Using the cDNA as a template, PCR was performed using primers designed based on the mRNA sequence information of human prothrombin, to prepare a polynucleotide encoding the full length or desired portion of prothrombin. The primer used in PCR can be appropriately designed based on the nucleotide sequence shown in SEQ ID NO.2, the human prothrombin sequence information registered in a database such as GenBank, or the like. Alternatively, a polynucleotide encoding a desired polypeptide can also be prepared by a conventional method using a commercially available nucleic acid synthesizer. Since codons encoding the respective amino acids are known, the nucleotide sequence of a polynucleotide encoding the amino acid sequence can be determined as long as the amino acid sequence can be determined. Then, the prepared polynucleotide is recombined into an appropriate vector, the polypeptide is expressed by an appropriate expression system, and the polypeptide is recovered, whereby a desired polypeptide can be obtained. The vector or various expression systems (bacterial expression system, yeast cell expression system, mammalian cell expression system, insect cell expression system, cell-free expression system, etc.) used are also known, and various vectors or host cells, reagents, and kits are commercially available, and therefore those skilled in the art can appropriately select and use them. Cultured human-derived cells are also commercially available, commonly assigned, and readily available.

In the present invention, an immunoassay for PIVKA-II is typically performed by a sandwich method. Sandwich immunoassays are well known per se as conventional methods. Specific examples thereof include various methods such as chemiluminescence Enzyme immunoassay (CLEIA), Enzyme-linked immunosorbent Assay (ELISA), radioimmunoassay, and electrochemiluminescence immunoassay.

In a sandwich assay system, one of the 2 antibodies or antigen-binding fragments thereof is usually used as a solid phase antibody immobilized on a solid phase, and the other is used as a labeled antibody labeled with a labeling substance. In the present invention, a mixture of the 1 st and 2 nd anti-prothrombin antibodies or antigen-binding fragments thereof is used as 1 antibody, and an anti-PIVKA-II antibody or antigen-binding fragment thereof is used as another 1 antibody, and any of them may be used as a solid phase antibody. In the following examples, the anti-PIVKA-II antibody was used as a solid phase antibody and an antibody mixture was used as a labeled antibody, but the present invention is not limited thereto.

The PIVKA-II measurement method of the present invention will be specifically described with reference to a case where a mixture of the 1 st and 2 nd anti-prothrombin antibodies is used as a labeled antibody. Of course, the mixture may be used as a solid phase antibody and an anti-PIVKA-II antibody as a labeled antibody, and the scope of the present invention is not limited to the specific examples described below. Instead of the antibody, an antigen-binding fragment of each antibody may be used.

First, the PIVKA-II antibody (solid phase antibody) was immobilized on a carrier. The solid-phase antibody is specifically bound to PIVKA-II by bringing the immobilized PIVKA-II antibody into contact with PIVKA-II contained in the subject. Then, after the carrier is washed with an appropriate buffer solution to remove components in the unbound sample, a mixture of the labeled substance 1 st and 2 nd anti-prothrombin antibodies (i.e., a mixture of at least 1 labeled 1 st and at least 1 labeled 2 nd anti-prothrombin antibodies) is bound to the PIVKA-II bound to the solid phase antibody. After the reaction is completed, the carrier is washed with, for example, an appropriate buffer solution to remove unreacted components, and then a signal from the labeling substance is detected by an appropriate method, whereby PIVKA-II contained in the sample can be measured.

The solid phase is not particularly limited, and may be the same as that used in a known sandwich immunoassay system. Specific examples of the solid phase material include: polystyrene, polyethylene, agarose, etc., but are not limited thereto. The physical shape of the solid phase is not essential. The solid phase used is preferably a material which facilitates the immobilization of the antibody on the surface of the solid phase and which can easily separate the immune complex formed in the measurement from unreacted components. Particularly preferred are plastic plates or magnetic particles used in conventional immunoassays. From the viewpoint of ease of handling, storage, and separation, it is most preferable to use magnetic particles made of the above-described material. The binding of the antibody to these solid phases can be carried out by conventional methods well known to those skilled in the art. The antibody may be bound to the solid phase by physical adsorption or by covalent bonding. For example, the glutaraldehyde method, periodic acid method, maleimide method, dipyridyl disulfide method, and the like can be used. Alternatively, antibody-bound particles can be obtained by adding an appropriate amount of antibody to a particle suspension in which magnetic particles are added to and dispersed in a buffer solution, stirring at 20 to 37 ℃ for about 1 hour, magnetically collecting the magnetic particles, and washing the particles with an appropriate buffer solution. The composition of the buffer solution to be used may be a composition which is conventional for immobilization of an antibody, and the pH may be in a range where the antibody is stably present and immobilization onto a solid phase is not inhibited.

The labeling substance is not particularly limited, and the same labeling substance as used in a known immunoassay system can be used, and specific examples thereof include, but are not limited to, enzymes, fluorescent substances, chemiluminescent substances, staining substances, radioactive substances, and the like.

When an enzyme is used as a labeling substance, a substrate such as a chromogenic substrate, a fluorogenic substrate or a luminescent substrate corresponding to the enzyme is reacted with the enzyme, and a signal generated therefrom is measured, whereby the enzyme activity is determined, and the measurement of the object to be measured is realized. For example, when ALP is used as the labeling substance, a luminescent substrate such as disodium 3- (4-methoxyspiro (1, 2-dioxetane-3, 2' -tricyclo [3.3.1.13,7] decane) -4-yl) phenylphosphate (for example, trade name AMPPD) can be used.

When biotin or hapten is used as the labeling substance, measurement can be performed using streptavidin or a hapten antibody to which an enzyme, a fluorescent substance, a chemiluminescent substance, a dye substance, a radioactive substance, or the like is bonded.

The detection of the signal may be appropriately selected depending on the kind of the labeling substance. For example, if the signal is a color, a colorimeter or an absorptiometer may be used, if the signal is fluorescence, a fluorometer may be used, if the signal is luminescence, a photon counter may be used, and if the signal is radiation, a radiation measuring device may be used. With respect to a standard sample containing PIVKA-II at a known concentration at various concentrations, PIVKA-II is measured by the method of the present invention, a standard curve is prepared by plotting the correlation between the signal value from the marker and the concentration of PIVKA-II in the standard sample, a sample having an unknown PIVKA-II concentration is subjected to the same measurement operation, the signal value from the marker is measured, and the measured signal value is substituted into the standard curve, whereby PIVKA-II in the sample can be quantified.

The subject to which the measurement method of the present invention is applied is a subject isolated from a subject, preferably a blood subject, and particularly preferably plasma or serum. The sample can be diluted as necessary and used.

The subject is not particularly limited as long as it is a mammal, and is usually a human, and may be a hepatocellular carcinoma patient or a hepatocellular carcinoma suspected patient, for example.

As described above, the 1 st and 2 nd anti-prothrombin antibodies used in the present invention for the immunoassay of PIVKA-II are at least 1 anti-prothrombin antibody recognizing a hydrophilic PIVKA-II molecule and at least 1 anti-prothrombin antibody recognizing a hydrophobic PIVKA-II molecule, respectively. In order to examine which of hydrophilic and hydrophobic PIVKA-II molecules a known antithrombin antibody or a newly prepared antithrombin antibody has affinity, each fraction obtained by fractionating a PIVKA-II-containing sample by hydrophobic interaction chromatography was used as a sample for measurement, and sandwich immunoassay using an antithrombin antibody and an anti-PIVKA-II antibody was carried out to examine which fraction has high reactivity.

Examples of the PIVKA-II-containing sample used for preparing the measurement sample include serum derived from a hepatocellular carcinoma patient. In the hydrophobic interaction chromatography, a hydrophobic column having a phenyl group as a functional group may be used, and elution may be performed using a linear concentration gradient of ammonium sulfate (e.g., 1.0M → 0M). About 20 to 30 elution fractions were collected, and the obtained 1 group of elution fractions were used as samples for measurement. When a plurality of patient-derived serums are used as the PIVKA-II-containing sample, a plurality of sets of measurement samples may be prepared by separately loading the serum samples into a chromatogram, or a mixture obtained by mixing a plurality of serum samples may be loaded into a chromatogram to prepare a measurement sample.

Each fraction of the prepared sample for measurement was measured by sandwich immunoassay using an antithrombin antibody and an anti-PIVKA-II antibody to be examined for affinity to hydrophobic and hydrophilic PIVKA-II molecules. The sandwich immunoassay system in this case can be constructed in the same manner as when the PIVKA-II immunoassay system is finally constructed. That is, if a PIVKA-II immunoassay system is constructed in which a mixture of the 1 st and 2 nd anti-prothrombin antibodies is used as a labeled antibody and an anti-PIVKA-II antibody is used as a solid phase antibody, the measurement of the measurement sample can be performed using the anti-prothrombin antibody to be examined for the affinity to hydrophobic and hydrophilic PIVKA-II molecules as the labeled antibody and the anti-PIVKA-II antibody as the solid phase antibody.

The elution fraction having a high ammonium sulfate concentration contains hydrophobic (hydrophilic) PIVKA-II molecules, and the elution fraction having a low ammonium sulfate concentration contains hydrophobic (hydrophobic) PIVKA-II molecules. In the present invention, the former elution fraction is referred to as PIVKA-II hydrophilic fraction, and the latter elution fraction is referred to as PIVKA-II hydrophobic fraction. By investigating the reactivity of these eluted fractions with an anti-prothrombin antibody, it was possible to investigate which of the hydrophilic and hydrophobic PIVKA-II molecules the anti-prothrombin antibody has an affinity for (recognizes).

The ammonium concentration in the hydrophilic and hydrophobic fractions separating PIVKA-II is usually selected from the range of 270mM to 370mM, for example from the range of 290mM to 350mM, 300mM to 340mM, 310mM to 330mM, 280mM to 330mM, or 290mM to 330 mM. The elution fraction having an ammonium sulfate concentration selected from these ranges is a hydrophilic fraction of PIVKA-II, and the elution fraction having a concentration lower than the ammonium sulfate concentration is a hydrophobic fraction of PIVKA-II.

In order to prepare a hydrophilic fraction and a hydrophobic fraction of PIVKA-II to be used for investigating affinity of an antithrombin antibody, at least 1 sample containing both hydrophilic PIVKA-II molecules and hydrophobic PIVKA-II molecules, or at least 2 samples including at least 1 sample containing at least hydrophilic PIVKA-II molecules and at least 1 sample containing at least hydrophobic PIVKA-II molecules are required. Whether any of the test subjects contained a hydrophilic PIVKA-II molecule and a hydrophobic PIVKA-II molecule can be examined by, for example, sandwich immunoassay using an anti-PIVKA-II antibody and an antithrombin polyclonal antibody. When a test sample containing both hydrophilic and hydrophobic PIVKA-II molecules is not obtained, a plurality of test samples can be used to prepare a measurement sample as described above.

After investigating the reactivity with the hydrophilic fraction and the hydrophobic fraction, the reactivity with each fraction (intensity of the detected signal) was plotted with the ammonium sulfate concentration on the horizontal axis and the signal value obtained by immunoassay on the vertical axis. The signal values may be plotted directly or by calculating the ratio of the signal intensity of each elution fraction to the total activity. When the reactivity with the hydrophilic fraction is high, the antithrombin antibody is judged to be an antibody having affinity with the hydrophilic PIVKA-II molecule. When the reactivity with the hydrophobic fraction is high, the antithrombin antibody is judged to be an antibody having affinity with the hydrophobic PIVKA-II molecule.

The mixing ratio of the 1 st antibody and the 2 nd antibody is not particularly limited as long as it is within a range in which both the hydrophobic PIVKA-II molecule and the hydrophilic PIVKA-II molecule in the sample can be measured, and the peak height or the peak area of the signal value of each fraction containing the PIVKA-II sample or the ratio of the signal intensity of each elution fraction to the total activity, which is plotted as described above, may be set to 1: 10-10: 1, e.g. 1: 5-5: range of 1, 1: 3-3: 1. or 1: 2-2: 1, in the above range. The weight ratio of the 1 st antibody to the 2 nd antibody (when a plurality of antibodies are used, the ratio of the total weight of the 1 st antibody to the total weight of the 2 nd antibody) can be set within a range in which both the hydrophobic PIVKA-II molecule and the hydrophilic PIVKA-II molecule in the sample can be measured, depending on the affinity of each antibody for PIVKA-II. The preferred mixing weight ratio of the 1 st antibody and the 2 nd antibody may vary depending on the height of the affinity of the 1 st antibody for hydrophilic PIVKA-II molecules and the height of the affinity of the 2 nd antibody for hydrophobic PIVKA-II molecules, and is not particularly limited, and in many cases, the mixing weight ratio of the antibodies may be set to 0.03: 1-30: 1, for example, 0.05: 1-20: range of 1, 0.1: 1-10: 1, or 0.3: 1-3: 1 to achieve a peak height or peak area in the above range. By combining at least 1 antithrombin antibody (antibody 1) having an affinity for a hydrophilic PIVKA-II molecule and recognizing the molecule and at least 1 antithrombin antibody (antibody 2) having an affinity for a hydrophobic PIVKA-II molecule and recognizing the molecule, and using a mixture of the two antibodies as a solid phase antibody or a labeled antibody, various molecular species of PIVKA-II present in a sample can be measured with high accuracy.

The PIVKA-II immunoassay reagent or kit for carrying out the PIVKA-II assay method of the present invention can be produced by the following steps.

(step 1) antibodies (antithrombin antibodies) or antigen-binding fragments thereof that bind to both PIVKA-II and prothrombin but do not exhibit reactivity with thrombin were examined for their reactivity to hydrophilic and hydrophobic fractions of PIVKA-II.

(step 2) mixing at least 1 antibody or antigen-binding fragment thereof reactive with the hydrophilic fraction and at least 1 antibody or antigen-binding fragment thereof reactive with the hydrophobic fraction.

The anti-prothrombin antibody or the antigen-binding fragment thereof used in step 1 may be a known anti-prothrombin antibody or an antigen-binding fragment thereof, or an anti-prothrombin antibody or an antigen-binding fragment thereof newly prepared as an antibody that binds to both PIVKA-II and prothrombin but does not react with thrombin. The method of investigating the reactivity of the hydrophilic and hydrophobic fractions of PIVKA-II is as described above.

The antibodies reactive with the hydrophilic fraction of PIVKA-II and the antibodies reactive with the hydrophobic fraction of PIVKA-II correspond to the antibodies referred to as the 1 st and 2 nd anti-prothrombin antibodies in the present specification, respectively. The mixture of at least 1 of the 1 st anti-prothrombin antibody or antigen-binding fragment thereof and at least 1 of the 2 nd anti-prothrombin antibody or antigen-binding fragment thereof may be provided in the form of a labeled substance bound thereto or immobilized on a solid phase such as a plastic plate or magnetic particles to prepare an immunoassay reagent or kit. Therefore, the method for producing the immunoassay reagent or kit may further comprise: a step of mixing the antithrombin antibody or the antigen-binding fragment thereof with the 1 st antibody and the 2 nd antibody or the antigen-binding fragment thereof before the step 1 or after the step 1 or in the step 2, and labeling the mixture with a labeling substance; alternatively, the step of immobilizing the solid phase after the step 2. When the anti-prothrombin antibody or an antigen-binding fragment thereof is immobilized on particles such as magnetic particles as a solid phase, the step of immobilizing on the solid phase may be performed before the step 1 or after the step 1, and the particles to which the antibodies are immobilized may be mixed in the step 2.

The immunoassay reagent produced by the above method may contain only a mixture of the 1 st antibody and the 2 nd antibody or an antigen-binding fragment thereof, may be in a form in which the mixture is dissolved in an appropriate buffer solution, and may further contain a component useful for stabilization of the antibodies and the like.

The immunoassay reagent may be provided as an immunoassay reagent or kit in combination with an anti-PIVKA-II antibody or an antigen-binding fragment thereof. For example, an immunoassay kit for PIVKA-II can be provided by combining a mixture of labeled 1 st and 2 nd anti-prothrombin antibodies or antigen-binding fragments thereof with a solid phase on which an anti-PIVKA-II antibody or antigen-binding fragment thereof is immobilized (or a combination of an anti-PIVKA-II antibody or antigen-binding fragment thereof and a solid phase for immobilizing the same). The labeled antibody and the immobilized antibody may be the reverse of those described above. The immunoassay kit may further contain a substrate for a labeling substance, a subject diluent, a washing solution, and the like.

Examples

The present invention is described in more detail below based on examples. Of course, the present invention is not limited to the following examples.

1. Hydrophobic interaction chromatography

A total of 1.0mL of a sample obtained by mixing 25 to 50. mu.L of a serum specimen with 500. mu.L of a 0.1M Phosphate Buffer (PB) containing 2M ammonium sulfate, pH7.0 (5.00 g/L potassium monohydrogen phosphate, 22.6g/L sodium dihydrogen phosphate (12 water salt), 264g/L ammonium sulfate; adjusted to pH7.0 by adding an appropriate amount of 4N sodium hydroxide) and 450. mu.L to 475. mu.L of a 0.1M PB pH7.0 (5.00 g/L potassium monohydrogen phosphate, 22.6g/L sodium dihydrogen phosphate (12 water salt), adjusted to pH7.0 by adding an appropriate amount of 4N sodium hydroxide) was subjected to chromatographic separation.

The column used in the hydrophobic interaction chromatography was HiTrap Phenyl HP (GE HEALTHCARE: single volume 1mL) and the apparatus was fractionated using AKTA FPLC UPC900(GE HEALTHCARE).

The column was equilibrated with 0.1M PB containing 1M ammonium sulfate and having a pH of 7.0 which was 10 times or more the column capacity, and 1.0mL of the sample was injected into the column and fractionated at a flow rate of 0.5 mL. The eluted fractions were recovered every 0.5 ml. The elution was carried out by using a linear gradient of salt concentration using 2 liquids of 0.1M PB pH7.0 (potassium monohydrogen phosphate 5.00g/L, sodium dihydrogen phosphate (12 water salt) 22.6g/L, ammonium sulfate 132 g/L) containing 1M ammonium sulfate, adjusted to pH7.0 by adding an appropriate amount of 4N sodium hydroxide, and 0.1M PB pH7.0 (potassium monohydrogen phosphate 5.00g/L, sodium dihydrogen phosphate (12 water salt) 22.6 g/L; pH adjustment was the same as above), and the ammonium sulfate concentration was changed from 1.0M to 0M during 30mL of elution.

2. Preparation of labeled antibody

Labeled antibodies were prepared using 4 kinds of monoclonal antibodies (antibody A, antibody B, antibody C, and antibody D) that bind to both PIVKA-II and prothrombin.

6mL of 3mg/mL antibody A solution was added to a G-25 column (manufactured by Framex) equilibrated with 0.1M citric acid buffer (pH3.5) to replace the antibody solution with the buffer. To this was added about 100. mu.L of a 1mg/mL pepsin solution, left at 37 ℃ for 1 hour, adjusted to a pH around neutral with a Tris buffer, and then added to a Superdex200 column (manufactured by Framesia) to perform gel filtration purification of the antibody. The single peaks at an absorbance of 280nm in the obtained fractions were combined as F (ab')₂And (3) fragment. In the F (ab')₂To 4mL of the fragment solution, 200. mu.L of a 0.2M solution of 2-mercaptoethylamine (hereinafter referred to as 2-MEA) was added, and the mixture was left at 37 ℃ for 2 hours to be subjected to reduction treatment. This was added to a G-25 column and the 2-MEA removed as a Fab' fragment of antibody A.

1.5mL of a high specific activity ALP solution of 10mg/mL was added to a G-25 column equilibrated with 0.1M phosphate buffer (pH7.0) to carry out buffer exchange of the ALP solution. To this was added 70. mu.L of a 10mg/mL solution of N- (4-maleimidobutyloxy) -succinimide (hereinafter, GMBS) in dimethylformamide, and the mixture was left at 30 ℃ for 1 hour to effect a reaction. This solution was added to a G-25 column equilibrated with 0.1M phosphate buffer (pH6.3) to remove excess GMBS, thereby preparing maleimido ALP. 4mL of the Fab' fragment solution of antibody A prepared above, 3mL of the maleimide ALP solution, and 13mL of 0.1M phosphate buffer (pH6.3) were mixed and left at room temperature for 1 hour to prepare an ALP-labeled antibody. 1mL of 2M 2-MEA solution was added thereto, the mixture was left at room temperature for 30 minutes, and the excess maleimide groups were blocked and then added to a Superdex200 column for purification. Fab' among several peaks at an absorbance of 280nm was combined with a peak having a molecular weight of 1:1 in ALP to obtain a purified ALP-labeled antibody A.

The labeled antibodies were also prepared for antibody B, antibody C and antibody D in the same manner.

Determination of PIVKA-II

A sample (e.g., a serum specimen) containing PIVKA-II was fractionated by hydrophobic interaction chromatography as described in the above item 1, and the obtained fractions were used as measurement samples. In the measurement, reagents (antibody-binding particles, PIVKA-II キャリブレータセット) attached to Lumipulse presto (registered trademark) PIVKA-II Eisai (manufactured by Fujiriebiio Co., Ltd.) and reagents (substrate solution, washing solution, etc.) (manufactured by Fujiriebeio Co., Ltd.) for Lumipulse presto were used in addition to the enzyme-labeled antibody.

First, 50. mu.L of antibody-binding particles (anti-PIVKA-II monoclonal antibody (mouse) binding ferrite particles) to which an anti-PIVKA-II monoclonal antibody (mouse) specifically binding to PIVKA-II was bound, which was carried by Lumipulse presto-PIVKA-II Eisai, was added with 20. mu.L of a sample for measurement, and the mixture was stirred and reacted at 37 ℃ for 8 minutes. The reaction residue was separated from the magnetic particles by magnetic force and washed with a washing solution. ALP-labeled antibody A (final concentration 0.36. mu.g/mL), antibody B (final concentration 0.5. mu.g/mL), antibody C (final concentration 0.12. mu.g/mL), and antibody D (final concentration 0.5. mu.g/mL), or the ALP-labeled anti-prothrombin polyclonal antibody attached to Lumipulse presto PIVKA-II Eisai, were added to the washed particles, and the mixture was stirred and reacted at 37 ℃ for 8 minutes. And separating the magnetic particles from the reaction residual liquid by magnetic force again, removing the reaction residual liquid, and cleaning by using a cleaning solution. To the particles, 200. mu.L of a substrate solution containing 3- (2 '-spiroadamantane) -4-methoxy-4- (3' -phosphoryloxy) phenyl-1, 2-dioxetane disodium salt (AMPPD), which is a chemiluminescent substrate for alkaline phosphatase, was added and reacted at 37 ℃ for 4 minutes. The amount of chemiluminescence (wavelength 463nm) after the reaction was measured with a luminometer. For the measurement, Lumipulse prestoiI (manufactured by Fujirebio) was used as a full-automatic chemiluminescence immunoassay apparatus.

FIG. 1 shows the results of measurement using 6 samples of serum samples from hepatocellular carcinoma patients, each fraction being fractionated by hydrophobic interaction chromatography, as a measurement sample, and using ALP-labeled antithrombin polyclonal antibody attached to Lumipulse presto PIVKA-II Eisai as an enzyme-labeled antibody. From the signal values of the respective fractions, activity values (mAU/mL) were calculated by the method described in the accompanying description of Lumipulse presto PIVKA-II Eisai, and the values were plotted. Some of the subjects have a subject forming a double peak and a subject forming a single peak, and the peak patterns are various. This indicates that: PIVKA-II in blood is a mixture of a plurality of molecules having different properties, i.e., a molecule having a higher hydrophilicity and a molecule having a higher hydrophobicity, and the ratio of each molecule varies depending on the blood sample. These hydrophilic and hydrophobic fractions were both PIVKA-II molecules, thus indicating: in order to measure PIVKA-II in blood accurately with good reproducibility, it is necessary to select a monoclonal antibody capable of reacting with both antibodies.

FIGS. 2-1 to 2-3 show the results of measurement using labeled antibodies A to D as measurement samples, each of 3 samples (sample No.4, sample No.269, and sample No.275) of serum samples from hepatocellular carcinoma patients, which were fractionated by hydrophobic interaction chromatography. From the signal values of the respective fractions, the ratio of the signal intensity of each fraction to the total activity (total signal value) to be detected was calculated, and the values were graphed. Antibody a and antibody B reacted with the hydrophilic fraction, and antibody C reacted with the hydrophobic fraction. Antibody D reacts primarily with the hydrophilic fraction and also reacts with the hydrophobic fraction. These results suggest that PIVKA-II in blood can be accurately measured by combining an antibody reactive with the hydrophilic fraction and an antibody reactive with the hydrophobic fraction.

Therefore, a mixture of antibodies (antibody a, antibody B, and antibody D) that reacted with the hydrophilic fraction of PIVKA-II and antibodies (antibody C) that reacted with the hydrophobic fraction was actually used as labeled antibodies, and the measurement samples prepared in 3 examples of the serum specimen were measured. From the signal values of the respective fractions, the ratio of the signal intensity of each fraction to the total activity detected was calculated, and the values were plotted. ALP-labeled antibody C (final concentration 0.12. mu.g/mL) was mixed with ALP-labeled antibody A (final concentration 0.36. mu.g/mL), antibody B (final concentration 0.5. mu.g/mL) and antibody D (final concentration 0.5. mu.g/mL) to prepare labeled antibodies. The measurement results are shown in FIGS. 3-1 to 3-3. It was confirmed that: by using 2 kinds of antibodies, that is, an antibody reactive with the hydrophilic fraction and an antibody reactive with the hydrophobic fraction, both the hydrophilic fraction and the hydrophobic fraction can be reacted as in the case of the antithrombin polyclonal antibody.

4. Investigation of mixing ratio of antibody

Then, the mixing ratio of the antibody reactive with the hydrophilic fraction of PIVKA-II (antibody a) and the antibody reactive with the hydrophobic fraction (antibody C) was examined. PIVKA-II was measured in the same manner as described in "measurement of PIVKA-II" 3, except that ALP-labeled antibody A and ALP-labeled antibody C were mixed at the ratios shown in tables 1 and 2 below. Serum samples (No.275) from hepatocellular carcinoma patients were used as the samples. The test body No.275 was confirmed to contain both hydrophilic PIVKA-II molecules and hydrophobic PIVKA-II molecules from the reactivity with the anti-prothrombin polyclonal antibody (FIG. 1).

The measurement results are shown in FIGS. 4-1 and 4-2. The vertical axis represents the actually measured count value. As a result, it was confirmed that: both hydrophilic and hydrophobic fractions can be detected at all mixing weight ratios studied (0.03: 1 to 30: 1).

Further, the ratio of the peak areas of the hydrophilic fraction and the hydrophobic fraction was evaluated. In this example, the boundary between the hydrophilic fraction and the hydrophobic fraction was set between ammonium sulfate concentrations of 288mM and 305mM, and the count values of the fractions contained in the peaks were accumulated to determine the peak areas of the hydrophilic fraction and the hydrophobic fraction, respectively. The peak area was determined by accumulating 14 fractions for both the hydrophilic fraction and the hydrophobic fraction using the values from 525mM to 68mM before the rising edge of the peak. As the count value, a value obtained by subtracting the count value of the fraction other than the peak from each count value is used in order to remove the background.

The peak area ratios calculated for the respective mixing ratios are shown in tables 1 and 2. Regarding the peak area ratio of the hydrophilic fraction to the hydrophobic fraction, in the case where the antibody mixing ratio (weight ratio) was 0.03: 1-30: 1 is in the range of 1: 2.1-2.8: 1. in the antibody mixing ratio of 0.05: 1-20: 1 is in the range of 1: 2.1-2.5: 1. in the antibody mixing ratio of 0.1: 1-10: 1 is in the range of 1: 1.9-1.8: 1. in the antibody mixing ratio of 0.3: 1-3: 1 is in the range of 1: 1.7-1.2: 1. from the above, it was confirmed that both the hydrophilic fraction and the hydrophobic fraction can be accurately detected by using the antibody a and the antibody C in a mixture.

[ Table 1]

[ Table 2]

Sequence listing

<110> Fuji Ribo Co., Ltd (FUJIREBIO Inc.)

Alcohole corporation (EIDIA Co., Ltd.)

<120> method for measuring PIVKA-II and method for producing PIVKA-II immunoassay reagent or kit

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Met Ala His Val Arg Gly Leu Gln Leu Pro Gly Cys Leu Ala Leu Ala

1 5 10 15

Ala Leu Cys Ser Leu Val His Ser Gln His Val Phe Leu Ala Pro Gln

20 25 30

Gln Ala Arg Ser Leu Leu Gln Arg Val Arg Arg Ala Asn Thr Phe Leu

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Xaa Xaa Val Arg Lys Gly Asn Leu Xaa Arg Xaa Cys Val Xaa Xaa Thr

50 55 60

Cys Ser Tyr Xaa Xaa Ala Phe Xaa Ala Leu Xaa Ser Ser Thr Ala Thr

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Asp Val Phe Trp Ala Lys Tyr Thr Ala Cys Glu Thr Ala Arg Thr Pro

85 90 95

Arg Asp Lys Leu Ala Ala Cys Leu Glu Gly Asn Cys Ala Glu Gly Leu

100 105 110

Gly Thr Asn Tyr Arg Gly His Val Asn Ile Thr Arg Ser Gly Ile Glu

115 120 125

Cys Gln Leu Trp Arg Ser Arg Tyr Pro His Lys Pro Glu Ile Asn Ser

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Thr Thr His Pro Gly Ala Asp Leu Gln Glu Asn Phe Cys Arg Asn Pro

145 150 155 160

Asp Ser Ser Thr Thr Gly Pro Trp Cys Tyr Thr Thr Asp Pro Thr Val

165 170 175

Arg Arg Gln Glu Cys Ser Ile Pro Val Cys Gly Gln Asp Gln Val Thr

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Val Ala Met Thr Pro Arg Ser Glu Gly Ser Ser Val Asn Leu Ser Pro

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Pro Leu Glu Gln Cys Val Pro Asp Arg Gly Gln Gln Tyr Gln Gly Arg

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Leu Ala Val Thr Thr His Gly Leu Pro Cys Leu Ala Trp Ala Ser Ala

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Gln Ala Lys Ala Leu Ser Lys His Gln Asp Phe Asn Ser Ala Val Gln

245 250 255

Leu Val Glu Asn Phe Cys Arg Asn Pro Asp Gly Asp Glu Glu Gly Val

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Asn Tyr Cys Glu Glu Ala Val Glu Glu Glu Thr Gly Asp Gly Leu Asp

290 295 300

Glu Asp Ser Asp Arg Ala Ile Glu Gly Arg Thr Ala Thr Ser Glu Tyr

305 310 315 320

Gln Thr Phe Phe Asn Pro Arg Thr Phe Gly Ser Gly Glu Ala Asp Cys

325 330 335

Gly Leu Arg Pro Leu Phe Glu Lys Lys Ser Leu Glu Asp Lys Thr Glu

340 345 350

Arg Glu Leu Leu Glu Ser Tyr Ile Asp Gly Arg Ile Val Glu Gly Ser

355 360 365

Asp Ala Glu Ile Gly Met Ser Pro Trp Gln Val Met Leu Phe Arg Lys

370 375 380

Ser Pro Gln Glu Leu Leu Cys Gly Ala Ser Leu Ile Ser Asp Arg Trp

385 390 395 400

Val Leu Thr Ala Ala His Cys Leu Leu Tyr Pro Pro Trp Asp Lys Asn

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caacatgttc tgtgctggtt acaagcctga tgaagggaaa cgaggggatg cctgtgaagg 1740

tgacagtggg ggaccctttg tcatgaagag cccctttaac aaccgctggt atcaaatggg 1800

catcgtctca tggggtgaag gctgtgaccg ggatgggaaa tatggcttct acacacatgt 1860

gttccgcctg aagaagtgga tacagaaggt cattgatcag tttggagagt agggggccac 1920

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Claims

1. A method for measuring PIVKA-II, which comprises measuring PIVKA-II in a subject by immunoassay,

the immunoassay uses a mixture of at least 1 st antithrombin antibody or antigen-binding fragment thereof recognizing a hydrophilic PIVKA-II molecule and at least 1 nd antithrombin antibody or antigen-binding fragment thereof recognizing a hydrophobic PIVKA-II molecule, and an anti-PIVKA-II antibody or antigen-binding fragment thereof specifically binding to PIVKA-II,

hydrophilic PIVKA-II molecules are PIVKA-II molecules contained in fractions having a predetermined ammonium sulfate concentration or higher among elution fractions obtained by fractionating a sample containing PIVKA-II by hydrophobic interaction chromatography; hydrophobic PIVKA-II molecules are PIVKA-II molecules contained in fractions below the specified ammonium sulfate concentration in the elution fraction,

the hydrophobic interaction chromatography uses a hydrophobic interaction chromatography column with phenyl as a functional group and a linear concentration gradient of ammonium sulfate,

the predetermined ammonium sulfate concentration is a concentration selected from the range of 270mM to 370 mM.

2. The assay method according to claim 1, wherein,

the predetermined ammonium sulfate concentration is selected from the range of 290 mM-350 mM.

3. The assay method according to claim 1, wherein,

the 1 st anti-prothrombin antibody, the 2 nd anti-prothrombin antibody and the anti-PIVKA-II antibody are monoclonal antibodies.

4. The assay method according to claim 2, wherein,

5. An assay according to any one of claims 1 to 4, wherein the immunoassay is carried out by a sandwich method,

the sandwich method uses the mixture as a labeled antibody and the anti-PIVKA-II antibody or an antigen-binding fragment thereof as a solid phase antibody.

6. The assay method according to any one of claims 1 to 4, wherein,

the mixing ratio of at least 1 st anti-prothrombin antibody or antigen-binding fragment thereof to at least 12 nd anti-prothrombin antibody or antigen-binding fragment thereof in the mixture is set to the following range:

when each fraction obtained by fractionating a PIVKA-II-containing sample by hydrophobic interaction chromatography was used as a measurement sample for immunoassay, the peak height or the peak area ratio of the obtained measurement values was 1: 10-10: 1, in the above range.

7. The assay method according to claim 5, wherein,

8. The assay method according to any one of claims 1 to 4, wherein,

the sample is serum or plasma.

9. The method according to claim 5, wherein the sample is serum or plasma.

10. The method according to claim 6, wherein the sample is serum or plasma.

11. The method according to claim 7, wherein the sample is serum or plasma.

12. A method of making a PIVKA-II immunoassay reagent or kit, comprising:

a step of examining the reactivity of an antibody or an antigen-binding fragment thereof that binds to both PIVKA-II and prothrombin but does not exhibit reactivity with thrombin, with respect to a hydrophilic fraction and a hydrophobic fraction of PIVKA-II; and

a step of mixing at least 1 antibody or an antigen-binding fragment thereof that reacts with the hydrophilic fraction and at least 1 antibody or an antigen-binding fragment thereof that reacts with the hydrophobic fraction,

the hydrophilic fraction of PIVKA-II is a fraction having an ammonium sulfate concentration equal to or higher than a predetermined concentration in the elution fraction; the hydrophobic fraction of PIVKA-II is the fraction of the elution fraction having less than the specified ammonium sulfate concentration,

the eluted fractions were obtained by fractionating a sample containing PIVKA-II using a hydrophobic interaction chromatography column having a phenyl group as a functional group and a linear concentration gradient of ammonium sulfate,

13. The manufacturing method according to claim 12,

14. The manufacturing method according to claim 12 or 13,

the antibody that binds both PIVKA-II and prothrombin is a monoclonal antibody.

15. The manufacturing method according to claim 12 or 13,

the mixing ratio of at least 1 antibody or antigen-binding fragment thereof reactive with the hydrophilic fraction and at least 1 antibody or antigen-binding fragment thereof reactive with the hydrophobic fraction is set to the following range:

16. The manufacturing method according to claim 14,